CN114561345B - Preparation method of mesenchymal stem cell preparation capable of improving safety of large-scale culture - Google Patents
Preparation method of mesenchymal stem cell preparation capable of improving safety of large-scale culture Download PDFInfo
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Abstract
The invention belongs to the technical field of stem cells, and particularly relates to a preparation method of a mesenchymal stem cell preparation capable of improving the safety of large-scale culture. The invention mainly adds taurine in the in-vitro large-scale expansion process of the mesenchymal stem cells, and simultaneously closely observes the fusion rate of the cells to control the cell density, inhibits the cells from forming a large amount of extracellular matrixes in the large-scale culture process, and avoids cell molding or cell agglomeration, thereby improving the application safety of the mesenchymal stem cells in animals, and being suitable for the application of cell factory culture to clinical transformation.
Description
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to a preparation method of a mesenchymal stem cell preparation capable of improving the safety of large-scale culture.
Background
Mesenchymal stem cells (Mesenchymal stem cells, MSC) are a type of stem cells with self-renewal, proliferation and multidirectional differentiation potential, widely exist in umbilical cord, bone marrow, dental pulp, fat or placenta tissues, and have the advantages of easy collection, preservation and transportation, no rejection of foreign bodies, avoidance of ethical disputes, stimulation of tissue regeneration, regulation of immune functions and the like.
Cell factory culture techniques have been used for over thirty years abroad, and have become increasingly popular in China in the last decade. Cell Factory (Cell Factory) is a Cell culture device with exquisite design, and utilizes the maximum culture surface in the limited space, thereby saving a large amount of Factory space, and realizing the purpose of expanding productivity without any Factory transformation. The method can be used for large-scale culture of mesenchymal stem cells, is particularly suitable for adherent cells, does not change the dynamic conditions of cell growth when being amplified from laboratory scale, ensures that the amplified culture is simple and feasible, has low pollution risk, saves space, can effectively ensure the sterility of operation, furthest reduces the difference between batches, realizes the operation regulation, greatly reduces the labor intensity and the concentration, and realizes the large-scale cell culture.
Taurine has a chemical structural formula of H 2 N-CH 2 -CH 2 -SO 3 H is named as beta-aminoethanesulfonic acid or 2-aminoethanesulfonic acid, has a relative molecular mass of 125.15, is monoclinic prismatic rod-like white crystals, has a melting point of 305.11 ℃, is nontoxic, odorless, slightly acidic and stable to heat.
At present, the mesenchymal stem cells are cultured by mostly adopting fetal bovine serum and basic culture solution, and some parts of the mesenchymal stem cells are cultured by adopting serum-free culture medium, but the local density of the mesenchymal stem cells is too high in the culture process, so that cell adhesion occurs, the adhered cells are injected into a body to easily cause embolism, the safety of the mesenchymal stem cells is reduced, and the clinical transformation of the mesenchymal stem cells is seriously hindered.
At present, adhered cells are generally removed by adopting a sieving mode in the industry, but a large amount of cells are wasted in such operation, which is not beneficial to the development of the cell industry.
Disclosure of Invention
Aiming at the condition that the existing mesenchymal stem cells are easy to form cell moulds and agglomerates when the culture density of the mesenchymal stem cells in a cell factory is high, the application provides a method capable of reducing or avoiding cell adhesion and agglomerate formation, thereby establishing a preparation method of a mesenchymal stem cell preparation capable of improving the safety of large-scale culture.
The invention aims to provide a preparation method of a mesenchymal stem cell preparation capable of improving the safety of large-scale culture, wherein taurine is added into a culture solution in the process of large-scale culture of mesenchymal stem cells.
Preferably, the preparation method of the mesenchymal stem cell preparation capable of improving the safety of large-scale culture is provided, wherein the mesenchymal stem cells are P1-P5 generation cells.
Preferably, the preparation method of the mesenchymal stem cell preparation capable of improving the safety of large-scale culture has the culture time of 3 days for each generation of mesenchymal stem cells.
Preferably, the preparation method of the mesenchymal stem cell preparation capable of improving the safety of large-scale culture has the advantage that the taurine in the culture solution is added at the concentration of 10-20 mmol/L.
Preferably, in the preparation method of the mesenchymal stem cell preparation capable of improving the safety of large-scale culture, the concentration of taurine added in the culture solution is 10mmol/L when the P1-P3 generation mesenchymal stem cells are cultured.
Preferably, in the preparation method of the mesenchymal stem cell preparation capable of improving the safety of large-scale culture, the concentration of taurine added in the culture solution is 20mmol/L when the P4-P5 generation mesenchymal stem cells are cultured.
Preferably, the preparation method of the mesenchymal stem cell preparation capable of improving the safety of large-scale culture further comprises the step of observing the cell fusion degree in the process of large-scale culture of cells.
Preferably, the preparation method of the mesenchymal stem cell preparation capable of improving the safety of large-scale culture has the cell fusion degree of 80-85%.
Preferably, in the above method for producing a mesenchymal stem cell preparation capable of improving the safety of mass culture, the culture medium used for mass culture of mesenchymal stem cells is an a-MEM culture medium containing 10% by volume of fetal bovine serum.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention mainly adds taurine in the in-vitro large-scale expansion process of the mesenchymal stem cells, and closely observes the fusion rate of the cells to control the cell density, inhibit the cells from forming a large amount of extracellular matrixes in the large-scale culture process, and avoid cell molding or cell agglomeration, thereby improving the application safety of the mesenchymal stem cells in animals. The method is simple and economical, is easy to industrialize, is suitable for cell factory culture and is easy to be applied to clinical transformation.
2. According to the invention, taurine is added into the culture solution in the culture process of the large-scale cell-amplified P1-P5 generation mesenchymal stem cells, so that the stem property of the cells is improved, and the amount of extracellular matrix secreted by the cells is reduced, thereby reducing cell adhesion.
3. According to the invention, the fusion rate of cells is observed in the culture process of the large-scale cell-expanded P1-P5 generation mesenchymal stem cells, so that the reasonable cell density is controlled, the cell molding is avoided, and the generation of cell aggregates is avoided.
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FIG. 1 is a morphological photograph of a normal group of mesenchymal stem cells;
FIG. 2 is a morphological photograph of mesenchymal stem cells of a treatment group;
FIG. 3 is a photograph of a normal group of mesenchymal stem cells in a suspended state;
fig. 4 is a photograph of a mesenchymal stem cell of a treatment group in a suspended state.
Detailed Description
In order that those skilled in the art will better understand the technical scheme of the present invention, the present invention will be further described with reference to specific embodiments and drawings.
In the description of the present invention, unless otherwise specified, all reagents are commercially available and methods are conventional in the art. All cell cultures in all experiments described below were expanded in a 10-layer cell factory.
Example 1 (treatment group)
A preparation method of a mesenchymal stem cell preparation capable of improving the safety of large-scale culture comprises the following steps:
s1, resuscitating the P0 generation mesenchymal stem cells by adopting a water bath kettle at 37 ℃, centrifuging to obtain the P1 generation mesenchymal stem cells, adding a cell culture solution, shaking uniformly, and culturing in a cell culture box;
the cell culture solution in the step is a-MEM culture solution (gibco) containing 10% of fetal bovine serum by volume fraction, and the concentration of taurine in the cell culture solution is 20mmol/L;
s2, after culturing for 3 days, when the cells grow to reach the fusion degree of 80%, digesting the cells, centrifuging to obtain P2 generation mesenchymal stem cells, adding a cell culture solution, shaking uniformly, and culturing in a cell culture box; the cell culture solution used in this step is referred to as S1;
s3, after culturing for 3 days, when the cells grow to reach the fusion degree of 80%, digesting the cells, centrifuging to obtain P3 generation mesenchymal stem cells, adding a cell culture solution, shaking uniformly, and culturing in a cell culture box; the cell culture solution used in this step is referred to as S1;
s4, after 3 days of culture, digesting the cells when the cell growth reaches 80% of fusion degree, obtaining P4 generation mesenchymal stem cells after centrifugation, adding a cell culture solution, shaking uniformly, and then placing the cells in a cell culture box for culture; the cell culture solution used in this step is referred to as S1;
s5, after culturing for 3 days, when the cells grow until the fusion degree reaches 80%, observing the cell morphology, and the result is shown in FIG. 2.
Example 2
A preparation method of a mesenchymal stem cell preparation capable of improving the safety of large-scale culture comprises the following steps:
s1, resuscitating the P0 generation mesenchymal stem cells by adopting a water bath kettle at 37 ℃, centrifuging to obtain the P1 generation mesenchymal stem cells, adding a cell culture solution, shaking uniformly, and culturing in a cell culture box;
the cell culture solution in the step is a-MEM culture solution containing 10% of fetal bovine serum by volume fraction, and the concentration of taurine in the cell culture solution is 10mmol/L;
s2, after culturing for 3 days, digesting the cells until the fusion degree reaches 80%, and observing the cell morphology.
Example 3
A preparation method of a mesenchymal stem cell preparation capable of improving the safety of large-scale culture comprises the following steps:
s1, resuscitating the P0 generation mesenchymal stem cells by adopting a water bath kettle at 37 ℃, centrifuging to obtain the P1 generation mesenchymal stem cells, adding a cell culture solution, shaking uniformly, and culturing in a cell culture box;
the cell culture solution in the step is a-MEM culture solution containing 10% of fetal bovine serum by volume fraction, and the concentration of taurine in the cell culture solution is 10mmol/L;
s2, after culturing for 3 days, when the cells grow to reach the fusion degree of 80%, digesting the cells, centrifuging to obtain P2 generation mesenchymal stem cells, adding a cell culture solution, shaking uniformly, and culturing in a cell culture box; the cell culture solution used in this step is referred to as S1;
s3, after culturing for 3 days, when the cells grow to reach the fusion degree of 80%, digesting the cells, centrifuging to obtain P3 generation mesenchymal stem cells, adding a cell culture solution, shaking uniformly, and culturing in a cell culture box; the cell culture solution used in this step is referred to as S1;
s4, after 3 days of culture, digesting the cells when the cell growth reaches 80% of fusion degree, obtaining P4 generation mesenchymal stem cells after centrifugation, adding a cell culture solution, shaking uniformly, and then placing the cells in a cell culture box for culture;
the cell culture solution used in the step is a-MEM culture solution containing 10% of fetal bovine serum by volume fraction, and the concentration of taurine in the cell culture solution is 20mmol/L;
s5, after culturing for 3 days, when the cells grow to reach the fusion degree of 80%, digesting the cells, centrifuging to obtain P5 generation mesenchymal stem cells, adding a cell culture solution, shaking uniformly, and culturing in a cell culture box; after 3 days of culture, the cells were digested and observed for cell morphology when the cells grew to 85% confluence.
The cell culture solution used in the step is a-MEM culture solution containing 10% of fetal bovine serum by volume fraction, and the concentration of taurine in the cell culture solution is 20mmol/L.
Experimental example (Normal group)
A method for preparing a mesenchymal stem cell preparation capable of improving the safety of large-scale culture is basically the same as the operation of example 1, except that the cell culture solution is an a-MEM culture solution containing 10% by volume of fetal bovine serum, and taurine is not added.
Comparing the cell morphology of example 1 with that of experimental example, the results are shown in fig. 1-4, as in fig. 1, the cells of the normal group grow in a spindle shape and adhere to the wall, and conform to the basic morphology of mesenchymal stem cells; as shown in fig. 2, the cells of the treatment group grew in a spindle shape, grew in an adherent manner, conforming to the basic morphology of mesenchymal stem cells. And digesting the cells, centrifuging to obtain P5 generation mesenchymal stem cells, wherein the cell suspension state of the normal group is in a circular dispersion state as shown in a graph of fig. 3, the cell mass exists occasionally, the cell suspension state of the treated group is in a circular dispersion state as shown in a graph of fig. 4, and no obvious mass exists under the lens.
The results of the cell identification by the flow assay are shown in Table 1, and the results of the mesenchymal stem cell surface molecule detection before and after the treatment meet the requirements of the International cell therapy Association, and the CD73, CD90 and CD105 are higher than 95 percent, and the CD14, CD19, CD34, CD45 and HLA-DR are lower than 2 percent, which indicates that the surface molecule expression of the cells is supported after the treatment.
The results show that the method can overcome the problems of adhesion, agglomeration and the like in the process of culturing the mesenchymal stem cells and improve the safety of clinical application.
TABLE 1 cell surface molecule detection results
It should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and because the adopted step method is the same as the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (4)
1. The preparation method of the mesenchymal stem cell preparation capable of improving the safety of large-scale culture is characterized in that taurine is added into a cell culture solution in the process of large-scale culture of the mesenchymal stem cells;
the method for culturing the mesenchymal stem cells on a large scale comprises the following steps:
s1, recovering the P0 generation mesenchymal stem cells, centrifuging to obtain the P1 generation mesenchymal stem cells, adding a cell culture solution, shaking uniformly, and culturing in a cell culture box; the cell culture solution is an a-MEM culture solution containing 10% of fetal bovine serum by volume fraction, and the taurine concentration in the cell culture solution is 10mmol/L-20 mmol/L;
s2, observing the cell fusion degree in the large-scale cell culture process, digesting the cells when the cell fusion degree is 80-85%, centrifuging to obtain P2 generation mesenchymal stem cells, adding the cell culture solution, shaking uniformly, and culturing in a cell culture box;
s3, repeating the step S2 to obtain the P3-P5 generation mesenchymal stem cells.
2. The method for preparing a mesenchymal stem cell preparation for improving the safety of large-scale culture according to claim 1, wherein the culture time of each generation of mesenchymal stem cells is 3 days.
3. The method for preparing a mesenchymal stem cell preparation capable of improving the safety of large-scale culture according to claim 1, wherein the concentration of taurine added to the cell culture solution is 10mmol/L when the mesenchymal stem cells of the generation P1-P3 are cultured.
4. The method for preparing a mesenchymal stem cell preparation capable of improving the safety of large-scale culture according to claim 1, wherein the concentration of taurine added to the cell culture solution is 20mmol/L when the P4-P5-generation mesenchymal stem cells are cultured.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2013205112A1 (en) * | 2005-10-19 | 2013-05-16 | Cornell Research Foundation, Inc. | Influenza viruses able to infect canids, uses thereof |
| CN106754678A (en) * | 2016-12-24 | 2017-05-31 | 叶宗耀 | A kind of culture medium suitable for dental pulp stem cell in vitro culture and preparation method thereof |
| CN112522191A (en) * | 2020-12-18 | 2021-03-19 | 云南中科灵长类生物医学重点实验室 | Culture method of mesenchymal stem cells |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2013205112A1 (en) * | 2005-10-19 | 2013-05-16 | Cornell Research Foundation, Inc. | Influenza viruses able to infect canids, uses thereof |
| CN106754678A (en) * | 2016-12-24 | 2017-05-31 | 叶宗耀 | A kind of culture medium suitable for dental pulp stem cell in vitro culture and preparation method thereof |
| CN112522191A (en) * | 2020-12-18 | 2021-03-19 | 云南中科灵长类生物医学重点实验室 | Culture method of mesenchymal stem cells |
Non-Patent Citations (2)
| Title |
|---|
| Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture;Dr R. L. Mauck Ph.D. et al.;《OsteoArthritis and Cartilage 》;第14卷;第179-189页 * |
| 牛磺酸对大鼠视神经胶质细胞损伤修复的影响;张焕婷;《中国优秀硕士学位论文全文数据库医药卫生科技辑》(第04期);第E057-170页 * |
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